Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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HUMAN VF:GF-SPECIFIC ANTISENSE OLIGONUCLFOTIDES
BACKGROUND OF THE INVENTION
This invention relates to neovascularization and vascular
endothelial growth factor. More specifically, this invention
relates to oligonucleotides specific for vascular endothelial
growth factor nucleic acid and useful treatment of disorders that
are associated with neovascularization and angiogenesis.
Neovascular diseases of the retina such as diabetic
retinopathy, retinopathy of prematurity, and age-related macular
degeneration are a major cause of blindness in the United States
and the world, yet the biochemical events responsible for these
processes have not been fully elucidated.
Diabetic retinopathy is the leading cause of blindness among
working age adults (20-64) in the United States (Foster in_
Harrison's Principles ofInternal Medicine (Isselbacher et al., eds.) McGraw-
Hill, Inc., New York (1994) pp. 1994-1995). During the course
of diabetes mellitus, the retinal vessels undergo changes that
result in not only leaky vessels but also vessel drop out
resulting in retinal hypoxia. The effects of these complications
are hemorrhaging, "cotton wool" spots, retinal infarcts, and
neovascularization of the retina resulting in bleeding and_
retinal detachment. If left untreated, there is a 60o chance of
visual loss. Classic treatment for proliferative diabetic
retinopathy is panretinal laser photocoagulation (PRP). However,
complications can occur from panretinal laser photocoagulation
such as foveal burns, hemorrhaging, retinal detachment, and
choroidal vessel growth. Furthermore, other untoward effects of
this treatment are decreased peripheral vision, decreased night
vision, and changes in color perception (Am. J. Ophthalmol. (1976)
81:383-396; Ophthalmol. (1991) 98:741-840).
Thus, there is a need for a more effective treatment for
diabetic retinopathy.
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Retinopathy of prematurity (ROP) is a common cause of
blindness in children in the United States (Pierce et al. (1994)
Int. Ophth. Clinics 34:121-148) . Premature babies are exposed to
hyperoxic conditions after birth even without supplemental oxygen
because the partial pressure of oxygen
in utero is much lower than what is achieved when breathing normal
room air. This relative hyperoxia is necessary for their
survival yet can result in ROP. The blood vessels of the retina
cease to develop into the peripheral retina resulting in ischemia
and localized hypoxic conditions as the metabolic demands of the
developing retina increase. The resulting hypoxia stimulates the
subsequent neovascularization of the retina. This
neovascularization usually regresses but can lead to irreversible
vision loss. There are at least 10,000 new cases per year with
a worldwide estimate of 10 million total cases. At present,
there is no effective cure for ROP. Two therapeutic methods,
cryotherapy and laser therapy, have been used but are not
completely effective and themselves cause damage to the eye,
resulting in a reduction of vision (Pierce et al. (1994) Int. Ophth.
Clinics 34:121-148) . Many other antiangiogenic compounds have been
tested, but no inhibition in retinal neovascularization has been
reported (Smith et al. (1994) Invest. Ophthalmol. Vis. Sci. 35:1442; Foley
et al. (1994) Invest. Ophthalmol. Vis. Sci. 35:1442) . Thus, there is a
need for an effective treatment for ROP.
Age related macular degeneration is one of the leading
causes of blindness in older adults in the United States, and may
account for up to 30% of all bilateral blindness among Caucasian
Americans (Anonymous (1994 ) Prevent Blindness America) . This disease
is characterized by loss of central vision, usually in both eyes,
due to damage to retinal pigment epithelial cells which provide
physiological support to the light sensitive photoreceptor cells
of the retina. In most cases there is currently no effective
treatment. In approximately 20% of exudative cases that are
diagnosed early, laser treatment can prevent further loss of
vision; however, this effect is temporary (Bressler et al.,
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Principles and Practices of Ophthalmology ( eds . Albert and Jakobiac ), W. B.
Saunders Co., Philadelphia, PA) (1994) Vol. 2 pp. 834-852).
= Thus, there is a need for a-more effective and permanent
treatment for age related macular degeneration.
Ocular neovascularization is also the underlying pathology
in sickle cell retinopathy, neovascular glaucoma, retinal vein
occlusion, and other hypoxic diseases. These eye diseases as
well as other pathological states associated with
neovascularization (i.e., tumor growth, wound healing) appear to
have hypoxia as a common factor (Knighton et al. (1983) Science
221:1283-1285; Folkman et al. (1987) Science 235:442-446; Klagsbrun
et al. (1991) Ann. Rev. Physiol. 53:217-239; Miller _ et al. (1993)
Principles and Practice of Ophthalmology, W. B. Saunders, Philadelphia, pp.
760; and Aiello et al. (1994) New Eng. J. Med. 331:1480-1487) .
Moreover, retinal neovascularization has been hypothesized to be
the result of a "vasoformative factor" which is released by the
retina in response to hypoxia (Michaelson (1948) Trans. Ophthalmol.
Soc. U. K. 68:137-180; and Ashton et al. (1954) Br. J. Ophthalmol.
38:397-432). Recent experimental data show a high correlation
between vascular endothelial growth factor expression and retinal
neovascularization (Aiello et al. (1994) New Eng. J. Med. 331 : 14 8 0-
1487). Furthermore, elevated levels of vascular endothelial
growth factor have recently been found in vitreous from patients
with diabetes (Aiello et al., ibid.). Thus, this cytokine/growth
factor may play an important role in neovascularization-related
disease.
Vascular endothelial growth factor/vascular permeability
= factor (VEGF/VPF) is an endothelial cell-specific mitogen which
has recently been shown to be stimulated by hypoxia and required
for tumor angiogenesis (Senger et al. (1986) Cancer 46:5629-5632;
Kim et al. (1993) Nature 3 62 : 841- 844 ; Schweiki et al. (1992) Nature
359:843-845; Plate et al. (1992) Nature 359:845-848) . It is a 34-
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43 kD (with the predominant species at about 45 kD) dimeric,
disulfide-linked glycoprotein synthesized and secreted by a
variety of tumor and normal cells. In addition, cultured human
retinal cells such as pigment epithelial cells and pericytes have =
been demonstrated to secrete VEGF and to increase VEGF gene
expression in response to hypoxia (Adamis et al. (1993) Biochem.
Biophys. Res. Commut2. 193:631-638; Plouet et al. (1992) Invest.
Ophthalmol. Vis. Sci. 3 4: 9 0 0; Adami s et al.(1993) Invest. Ophthalmol.
Vis. Sci.
34:1440; Aiello et al. (1994) Invest. Ophthalmol. Vis. Sci. 35:1868;
Simorre-Pinatel et al.(1994) Invest. Ophthalmol. Vis. Sci. 35:3393-3400) .
In contrast, VEGF in normal tissues is relatively low. Thus,
VEGF appears to play a principle role in many pathological states
and processes related to neovascularization. Regulation of VEGF
expression in tissues affected by the various conditions
described above could therefore be key in treatment or
preventative therapies associated with hypoxia.
New chemotherapeutic agents termed "antisense
oligonucleotides" have been developed which are capable of
modulating cellular and foreign gene expression (see, Zamecnik
et al. (1978) Proc. Natl. Acad. Sci. (USA) 75:280-284) . Without being
limited to any theory or mechanism, it is generally believed that
the activity of antisense oligonucleotides depends on the binding
of the oligonucleotide to the target nucleic acid (e.g. to at
least a portion of a genomic region, gene or mRNA transcript
thereof), thus disrupting the function of the target, either by
hybridization arrest or by destruction of target RNA by RNase H
(the ability to activate RNase H when hybridized to RNA).
VEGF-specific antisense oligonucleotides have been developed
(Uchida et al. (1995) AntisenseRes. & Dev. 5 (1) :87 (Abstract OP-l0) ;
Nomura et al., (1995) Antisense Res. & Dev. 5 (1) :91 (Abstract OP-18 )),
although_ none have been demonstrated to reverse
neovascularization or angiogenesis. Thus, a need still remains
for the development of o_ligonucleotides that are capable of
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reducing VEGF expression, and ultimately, of inhibiting the onset
of diseases and disorders associated with the expression of VEGF.
SUMMARY OF THE INVENTION
It is known that cells affected by hypoxia induce VEGF. It
has now been discovered that synthetic oligonucleotides specific
for the mRNA for VEGF can inhibit hypoxia-associated
neovascularization. It has also been discovered that
oligonucleotides specific for nucleotides 58 to 90 of the VEGF
gene can reduce the hypoxia-induced expression of VEGF mRNA and
protein. This information has been exploited to develop the
present invention which includes VEGF-specific oligonucleotides,
pharmaceutical formulations, methods of reducing the expression
of VEGF mRNA and protein, and methods of reducing
neovascularization and of treating disorders and diseases related
to neovascularization. As used herein, the term
"neovascularization" refers to the growth of blood vessels and
capillaries.
In one aspect, the invention provides synthetic
oligonucleotides specific for human vascular endothelial growth
factor nucleic acid and effective in inhibiting the expression
of vascular endothelial growth factor is administered to a
neovascularized tissue. This tissue may be a culture or may be
part or the whole body of an animal such as a human or other
mammal. In one embodiment, this invention provides synthetic
oligonucleotides complementary to human VEGF-specific nucleic
acids, and having a nucleic acid sequence set forth in the
Sequence Listing as SEQ ID NOS:2-16.
As used herein, the term "synthetic oligonucleotide" refers
to chemically synthesized polymers of nucleotides covalently
attached via at least-one 5' to 3' internucleotide linkage. In
some embodiments, these oligonucleotides contain at least one
deoxyribonucleotide, ribonucleotide, or both deoxyribonucleotides
and ribonucleotides. In other embodiments, the synthetic
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oligonucleotides used in the methods of the invention are from
about 14 to about 28, or about 15 to about 30 nucleotides in
length. In some preferred embodiments, these oligonucleotides
contain from about 15 to about 25 nucleotides, and in other =
embodiments, from about 16 to 29 nucleotides.
For purposes of the invention, the term "oligonucleotide
sequence that is complementary to a genomic region or an RNA
molecule transcribed therefrom" is intended to mean an
oligonucleotide that binds to the nucleic acid sequence under
physiological conditions, e.g., by Watson-Crick base pairing
(interaction between oligonucleotide and single-stranded nucleic
acid) or by Hoogsteen base pairing (interaction between
oligonucleotide and double-stranded nucleic acid) or by any other
means including in the case of a oligonucleotide binding to RNA,
causing pseudoknot formation. Binding by Watson-Crick or
Hoogsteen base pairing under physiological conditions is measured
as a practical matter by observing interference with the function
of the nucleic acid sequence.
In some embodiments, the synthetic oligonucleotides of the
invention are modified in a number of ways without compromising
their ability to hybridize to nucleotide sequences contained
within the mRNA for VEGF. The term "modified oligonucleotide"
as used herein describes an oligonucleotide in which at least two
of its nucleotides are covalently linked via a synthetic linkage,
i.e., a linkage other than a phosphodiester linkage between the
5' end of one nucleotide and the 3' end of another nucleotide in
which the 5' nucleotide phosphate has been replaced with any
number of chemical groups.
In some preferred embodiments, at least one internucleotide
linkage of the oligonucleotide is an alkylphosphonate,
phosphorothioate, phosphorodithioate, phosphate ester,
alkylphosphonothioate, phosphoramidate, carbamate, carbonate,
phosphate triester, acetamidate, and/or carboxymethyl ester.
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The term "modified oligonucleotide" also encompasses
oligonucleotides having at least one nucleotide with a modified
base and/or sugar such as a 2'-O-substituted ribonucleotide. For
purposes of the invention, the term "2'-O-substituted" means
substitution of the 2' position of the pentose moiety with an -0-
lower alkyl group containing 1-6 saturated or unsaturated carbon
atoms, or with an -0-aryl or allyl group having 2-6 carbon atoms,
wherein such alkyl, aryl or allyl group may be unsubstituted or
may be substituted, e.g., with halo, hydroxy, trifluoromethyl,
cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or
amino groups; or with a hydroxy, an amino or a halo group, but
not with a 2'-H group. In some embodiments the oligonucleotides
of the invention include four or five ribonucleotides 2'-O-
alkylated at their 5' terminus (i.e., 5' 2-0-alkylated
ribonucleotides), and/or four or five ribonucleotides 2'-O-
alkylated at their 3' terminus (i.e., 3' 2-0-alkylated
ribonucleotides). In preferred embodiments, the nucleotides of
the synthetic oligonucleotides are linked by a or at least one
phosphorothioate internucleotide linkage. The phosphorothioate
linkages may be mixed Rp and SP enantiomers, or they may be
stereoregular or substantially stereoregular in either Rp or SP
form (see Iyer et al. (1995) Tetrahedron Asymmetry 6:1051-1054) .
In another aspect of the invention, a method of treating
retinopathy of prematurity (ROP) is provided. This method
comprises the step of administering to a subject afflicted with
ROP a therapeutic amount of an oligonucleotide specific for
vascular endothelial growth factor nucleic acid and effective in
inhibiting the expression of vascular endothelial growth factor
in the retina. In another aspect of the invention, a method of
treating diabetic retinopathy is provided. This method includes
administering to a subject afflicted with diabetic retinopathy
a therapeutic amount of an oligonucleotide specific for vascular
endothelial growth factor nucleic acid and effective in
inhibiting the expression of VEGF in the retina.
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In yet another aspect of the invention, a method of treating
age-related macular degeneration (ARMD) is provided, which
includes comprising the step of administering to a subject
afflicted with ARMD a therapeutic amount of an oligonucleotide
specific for vascular endothelial growth factor nucleic acid
effective in inhibiting the expression of VEGF in the retina.
Another aspect of the invention is assessment of the role
of VEGF in neovascularization and angiogenesis associated with
disease states.
In another aspect, the invention provides a method of
inhibiting VEGF expression. In this method, nucleic acid -
specific for VEGF is contacted with an oligonucleotide of the
invention. As used herein, the term "nucleic acid" encompasses
a genomic region or an RNA molecule transcribed therefrom. In
some embodiments, the nucleic acid is mRNA.
Without being limited to any theory or mvchanism, it is
generally believed that the activity of oligonucleotides used in
accordance with this invention depends on the hybridization of
the oligonucleotide to the target nucleic acid (e.g. to at least
a portion of a genomic region, gene or mRNA transcript thereof),
thus disrupting the function of the target. Such hybridization
under physiological conditions is measured as a practical matter
by observing interference with the function of the nucleic acid
sequence. Thus, a preferred oligonucleotide used in accordance
with the invention is capable of forming a stable duplex (or
triplex in the Hoogsteen pairing mechanism) with the target
nucleic acid; activate RNase H thereby causing effective
destruction of the target RNA molecule, and in addition is
capable of resisting nucleolytic degradation (e.g. endonuclease
and exonuclease activity) invivo. A number of the modifications
to oligonucleotides described above and others which are known
in the art specifically and successfully address each of these preferred
characteristics. Also provided by the present
invention is a pharmaceutical composition comprising at least one
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synthetic oligonucleotide complementary to a nucleic acid
specific for human vascular endothelial growth factor in a
physiologically acceptable carrier.
In some preferred embodiments of the methods of the
invention described above, the oligonucleotide is administered
locally (e.g., intraocularly or interlesionally) and/or
systemically. The term "local administration" refers to delivery
to a defined area or region of the body, while the term "systemic
administration is meant to encompass delivery to the whole
organism by oral ingestion, or by intramuscular, intravenous,
subcutaneous, or intraperitoneal injection.
Another aspect of the invention includes pharmaceutical
compositions capable of inhibiting neovascularization and thus
are useful in the methods of the invention. These compositions
include a synthetic oligonucleotide of the present invention
which specifically inhibits the expression of vascular
endothelial growth factor and a physiologically and/or
pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable" means a non-toxic
material that does not interfere with the effectiveness of the
biological activity of the active ingredient(s) . The term
"physiologically acceptable" refers to a non-toxic material that
is compatible with a biological system such as a cell, cell
culture, tissue, or organism.
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BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects of the present invention,
the various features thereof, as well as the invention itself may =
be more fully understood from the following description, when
read together with the accompanying drawings in which:
FIG. 1 is a diagrammatic representation of the murine model
for retinal neovascularization;
FIG. 2 is a graphic representation of the ability of
oligonucleotides of the invention to inhibit neovascularization
during retinopathy of prematurity;
FIG. 3 is a diagrammatic representation of the ELISA used
to test the ability of human VEGF-specific oligonucleotides to
inhibit the expression ofVEGF;
FIG. 4 is a graphic representation of the results of an
ELISA demonstrating the reduction in the expression of VEGF in
human cells in the presence of - human_ VEGF-specific
oligonucleotides of the invention;
FIG. 5 is a graphic representation of the results of a
Northern blot demonstrating the reduction in the expression of
VEGF by human cells in the presence of varying concentrations of
human VEGF-specific oligonucleotides of the invention;
FIG. 6 is a schematic representation of regions of the VEGF
cDNA sequence that are targeted by oligonucleotides of the
invention;
FIG. 7 is a graphic representation of ELISA results
demonstrating the ability of oligonucleotides H3-I and H3-J to
inhibit VEGF expression induced by cobalt chloride;
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FIG. 8 is a graphic representation of ELISA results
demonstrating the ability of oligonucleotides H3-D, H3-E, and H3-
F to inhibit VEGF expression induced by cobalt chloride;
FIG. 9 is a graphic representation of ELISA results
demonstrating the ability of oligonucleotides H3-G and H3-H to
inhibit VEGF expression induced by cobalt chloride;
FIG. 10 is a graphic representation of ELISA results
demonstrating the ability of oligonucleotides H3 and H3-I to
inhibit VEGF expression induced by cobalt chloride in M21 human
melanoma cells in vitro; and
FIG. 11 is a graphic representation of ELISA results
demonstrating the ability of modified H3 oligonucleotides to
inhibit VEGF expression induced by cobalt chloride (H3-K: all 2'-
0-methylated phosphorothioate ribonucleotides; H3-L: five 5' 2'-
0-alkylated phosphorothioate ribonucleotides, the= remainder,
phosphorothioate deoxyribonucleotides; H3-M: five 3' 2'-O-
alkylated phosphorothioate ribonucleotides, the remainder,
phosphorothioate deoxyribonucleotides; and H3-N: five 3' 2'-O-
alkylated phosphorothioate ribonucleotides, five 5' 2'-O-
alkylated phosphorothioate ribonucleotides, and the remainder,
phosphorothioate deoxyribonucleotides).
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill
in the art.
The present invention provides synthetic antisense
oligonucleotides specific for VEGF nucleic acid which are useful
in, treating diseases and disorders associated with
neovascularization and angiogenesis, including retinal
neovascularization.
Antisense oligonucleotide technology provides a novel
approach to the inhibition of gene expression (see generally,
Agrawal (1992) Trends in Biotech. 10 : 152 -158 ; Wagner (1994) Nature
372:333-335; and Stein et al. (1993) Science 261:1004-1012) . By
binding to the complementary nucleic acid sequence (the sense
strand) , antisense oligonucleotide are able to inhibit splicing
and translation of RNA. In this way, antisense oligonucleotides
are able to inhibit protein expression. Antisense
oligonucleotides have also been shown to bind to genomic DNA,
forming a triplex, and inhibit transcription. Furthermore, a
17mer base sequence statistically occurs only once in the human
genome, and thus extremely precise targeting of specific
sequences is possible with such antisense oligonucleotides.
It has been determined that the VEGF coding region is
comprised of eight exons (Tischer et al. (1994) J. Biol. Chem.
266:11947-11954). Three VEGF transcripts, 121, 165, and 189
amino acids long, have been observed, suggesting that an
alternative splicing mechanism is involved (Leung et al. (1989)
Science 246:1306-1309; Tischer et al. (1991) J.Biol. Chem. 266:11947-
11954). More recently, a fourth VEGF transcript was discovered
which has a length encoding 206 amino acids (Houck et al. (1991)
Mol. Endocrinol. 5:1806-1814) . Transcripts analogous to the 121 and
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165 amino acid polypeptides have been identified in the bovine
system (Leung et al. (1989) Science 246:1306-1309) , and the
transcript corresponding to the 165 amino acid transcript have
also been identified in the rodent system (Conn et al. (1990)
Proc. Natl. Acad. Sci. (USA) 87:1323-1327) ; Senger et al. (1990) Cancer
Res. 50:1774-1778; Claffey et al. (1992) J. Biol. Chem. 267:16317-
16322). Nucleic acid sequences encoding three forms of VEGF have
also been reported in humans (Tischer et al. (1991) J. Biol. Chem.
266:11947-11954), and comparisons between the human and the
murine VEGF have revealed greater than 85o interspecies
conservation (Claffey et al. (1992) J. Biol. Chem. 267:16317-16322) .
The oligonucleotides of the invention are directed to any
portion of the VEGF nucleic acid sequence thateffectively acts
as a target for inhibiting VEGF expression. The sequence of the
gene encoding VEGF has been reported in mice (Claffey et al.,
ibid.) and for humans (Tischer et al., ibid. ). These targeted
regions of the VEGF gene include any portions of the known exons.
In addition, exon-intron boundaries are potentially useful
targets for antisense inhibition of VEGF expression. The
nucleotide sequences of some representative, non-limiting
oligonucleotides specific for human VEGF are listed below in
TABLE 1.
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TABLE 1
SEQ
TARGETED ID
OLIGO SITE SEOUENCE (AS) NO:
H-1 _21-2 5'-CGCCGGGCCGCCAGCACACT-3' 1
H=1R 21-2 5'-CGCCGGGCCGCCAGCACACU-3' 2
H-lA 16-2 5'-GGCCGCCAGCACACT-3' 3 =
H-lB 26-2 5'-GCTCGCGCCGGGCCGCCAGCACACT-3' 4
H-2 76-57 5'-CAAGACAGCAGAAAGTTCAT-3' S
H-3 80-62 5'-CACCCAAGACAGCAGAAAG-3' 6
H-3A 80-66 5'-CACCCAAGACAGCAG-3' 7
H-3B 86-62 5'-CCAATGCACCCAAGACAGCAGAAAG-3' 8
H-4 64-45 5'-AAGTTCATGGTTTCGGAGGC-3' 10
H-5 62-43 5'-GTTCATGGTTTCGGAGGCCC-3' 11
H-6 138-119 5'-GTGCAGCCTGGGACCACTTG-3' 12
H-7 628-609 5'-CGCCTCGGCTTGTCACATCT-3' 13
H-8 648-629 5'-CTTCCTCCTGCC.CGGCTCAC-3' 14
H-8R 648-629 5'-CUUCCUCCUGCCCGGCUCAC-3' 15
H-8A 648-634 5'-CTTCCTCCTGCCCGG-3' 16
H-8B 653-629 5'-GGCTCCTTCCTCCTGCCCGGCTCAC-3' 17
H-9 798-779 5'-GTCTCCTCTTCCTTCATTTC-3' 18
H-9A 798-784 5'-GTCTCCTCTTCCTTC-3' 19
H-9B 803-779 S'-GCAGAGTCTCCTCTTCCTTCATTTC-3' 20
H-10 822-803 5'-CGGACCCAAAGTGCTCTGCG-3' 21
H-l0A 817-803 5'-CCAAAGTGCTCTGCG-3' 22
H-lOB 827-803 5'-CCCTCCGGACCCAAAGTGCTCTGCG-3' 23
H-il El-Il 5'-GGGCACGACCGCTTACCTTG-3' 24
H-12 Il-E2 5'-GGGACCACTGAGGACAGAAA-3' 25
H-13 I2-E3 5'-CACCACTGCATGAGAGGCGA-3' 26
H-14 E3-I3 5'-TCCCAAAGATGCCCACCTGC-3' 27
H-15 I3-E4 5'-CGCATAATCTGGAAAGGAAG-3' 28
H-17 59-40 5'-CATGGTTTCGGAGGCCCGAC-3' 30_
3S_ H-17B 59-40 5'-CAUGGTTUCGGAGGCCCGAC-3' 31
H-18 61-42 5'-TTCATGGTTTCGGAGGCCCG-3' 32
El/I1 El/Il 5'-GACCGCTTACCTTGGCATGG-3' 33
I1/E2 Il/E2 5'-CCTGGGACCACTGAGGACAG-3' 34
E2/I2 E2/I2 5'-GGGACTCACCTTCGTGATGA-3' 35
12/E3 12/E3 5'-GAACTTCACCACTGCATGAG-3' 36
E3/I3 E3/I3 5'-TCCCAAAGATGCCCACCTGC-3' 37
I3/E4 I3/E4 5'-GCATAATCTGGAAAGGAAGG-3' 38
E4/I4 E4/I4 S'-ACATCCTCACCTGCATTCAC-3' 39
E4/I4B E4/I4 5'-ACATCCUCACCTGCAUUCAC-3' 40
14/ES I4/E5 5'-TTTCTTTGGTCTGCAATGGG-3' 41
E5/I5 ES/IS 5'-GGCCACTTACTTTTCTTGTC-3' 42
I5/E7 IS/E7 5'-CACAGGGACTGGAAAATAAA-3' 43
E7/I7 E7/I7 S'-GGGAACCAACCTGCAAGTAC-3' 44
17/E8 17/E8 5'-GTCACATCTGAGGGAAATGG-3' 45
VH 641-621 5'-CTGCCCGGCTCACCGCCTCGG-3 46
H-19 56-38 5'-GGTTTCGGAGGCCCGACCG-3' 50
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With the published nucleic acict sequences and the disclosure
provided herein, those of skill in the art will be able to
identify, with without undue experimentation, other antisense
nucleic acid sequences that inhibit VEGF expression. For example,
S other sequences targeted specifically to human VEGF nucleic acid
can be selected based on their ability to be cleaved by RNAse H.
One useful targeted region is around bases 58 to 90. The
nucleotide sequences of some representative, non-limiting
oligonucleotides specific for human VEGF have SEQ ID NOS:54-68.
The oligonucleotides of the invention are composed of
ribonucleotides, deoxyribonucleotides, or a combination of both,
with the S' end of one nucleotide and the 3' end of another
nucleotide being covalently linked. These oligonucleotides are
at least 14 nucleotides in length, but are preferably 1.5 to 30
nucleotides long, with 15 to 29mers being the most common.
These oligonucleotides can be prepared by the art recognized
methods such as phosphoramidate or H-phosphonate chemistry which
can be -carried out manually or by an automated synthesizer as
described in Uhlmann et al. (Chem. Rev. (1990) 90:534-583) and
Agrawal (Trends Biotechnol. (1992) 10 :152 -158 )
The oligonucleotides of the invention may also be modified
in a number of ways without compromising their ability to
hybridize to VEGF mRNA. For example, the oligonucleotides may
contain at least one or a combination of other than
phosphodiester internucleotide linkages between the 5' end of one
nucleotide and the 3' end of another nucleotide in which the 5'
nucleotide phosphodiester linkage has been replaced with any
number of chemical groups. Examples of such chemical groups
include alkylphosphonates, phosphorothioates,
phosphorodithioates, alkylphosphonothioates, phosphoramidates,
phosphate esters, carbamates, acetamidate, carboxymethyl esters,
carbonates, and phosphate triesters.
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For example, U.S. Patent No. 5,149,797 describes traditional
chimeric oligonucleotides having a phosphorothioate core region
interposed between methylphosphonate or phosphoramidate flanking
regions.
Various oligonucleotides with
modified internucleotide linkages can be prepared according to
known methods ( see , e. g., Goodchild (1990 ) Bioconjugate Chem. 2 : 165 -
187; Agrawal et al. , (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083;
Uhlmann et al. (1990) Chem. Rev. 90 : 534-583 ; and Agrawal et al.
(1992) Trends Biotechnol. 10 : 15 2-15 8.
The phosphorothioate linkages may be mixed Rp and Sp
enantiomers, or they may be stereoregular or substantially
stereoregular in either Rp or Sp form (see Iyer et al. (1995)
Tetrahedron Asymmetry 6:1051-1054 ). Oligonucleotides with
phosphorothioate linkages can be prepared using methods well
known in the field such as phosphoramidite (see, e.g., Agrawal
et al. (1988) Proc. Natl. Acad. Sci. (USA) 8 5: 7 0 7 9- 7 0 8 3). or by H-
phosphonate (see, e.g. , Froehler (1986) Tetrahedron Lett. 27:5575-
5578) chemistry. The synthetic methods described in Bergot et
al. (J. Chromatog. (1992) 559:35-42) can also be used.
Oligonucleotides which are self-stabilized are also
considered to be modified oligonucleotides useful in the methods
of the invention (Tang et al. (1993) NucleicAcidsRes. 20:2729-2735) .
These oligonucleotides comprise two regions: a target
hybridizing region; and a self-complementary region having an
oligonucleotide sequence complementary to a nucleic acid sequence
that is within the self-stabilized oligonucleotide.
Other modifications include those which are internal or at
the end(s) of the oligonucleotide molecule'and include additions
CA 02214431 2005-02-28
-17-
to the molecule of the internucleoside phosphate linkages, such
as cholesteryl or diamine compounds with varying numbers of
carbon residues between the amino groups and terminal ribose,
deoxyribose and phosphate modifications which cleave, or
crosslink to the opposite chains or to associated enzymes or
other proteins which bind to the genome. Examples of such
modified oligonucleotides include oligonucleotides with a
modified base and/or sugar such as arabinose instead of ribose,
or a 3', 5'-substituted oligonucleotide having a sugar which, at
both its 3' and 5' positions is attached to a chemical group
other than a hydroxyl group (at its 3' position) and other than
a phosphate group (at its 5' position).
Other examples of modifications to sugars include
modifications to the 2' position of the ribose moiety which
include but are not limited to 2' -O-substituted with an -0- lower
alkyl group containing 1-6 saturated or unsaturated carbon atoms,
or with an -0-aryl, or allyl group having 2-6 carbon atoms
wherein such -0-alkyl, aryl or allyl group may be unsubstituted
or may be substituted, (e.g., with halo, hydroxy, trifluoromethyl
cyano, nitro acyl acyloxy, alkoxy, carboxy, carbalkoxyl, or amino
groups) , or with an amino, or halo group. None of these
substitutions are intended to exclude the native 2'-hydroxyl
group in the case of ribose or 2'-H- in the case of deoxyribose.
PCT Publication No. WO 94/02498 discloses traditional hybrid
oligonucleotides having regions of 2'-O-substituted
ribonucleotides flanking a DNA core region.
Nonlimiting examples of particularly useful oligonucleotides of
the invention have 2'-O-alkylated ribonucleotides at their 3',
5', or 3' and 5' termini, with at least four or five contiguous
nucleotides being so modified. Non-limiting examples of 2'-O-
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WO 96/27006 PCT/US96/02840
-18-
alkylated groups include 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl,
and 2'-O-butyls.
Other modified oligonucleotides are capped with a nuclease
resistance-confe-rring bulky substituent at their 3' and/or 5'
end(s) , or have a substitution in one nonbridging oxygen per
nucleotide. Such modifications can be at some or all of the
internucleoside linkages, as well as at either or both ends of
the oligonucleotide and/or in the interior of the molecule.
A nonlimiting list of useful unmodified and modified
oligonucleotides of the invention are listed below in Table 2.
TABLE 2
TARGETED SEQ ID
OLIGO SITE (5' -> 3') NO:
H-3lal 81-62 GCACCCAAGACAGCAGAAAG 55
H-31a2 81-62 GCACCCAAGACAGCAGAAAG 55
H-3Ia3 81-62 GCACCCAAGACAGCAGAAAG 55
H-31a4 81-62 GCACCCAAGACAGCAGAA.AG 55
H-3Ia5 81-62 GCACCCAAGACAGCAGAAAG 55
H-31a6 81-62 GCACCCAAGACAGCAGAAAG 55
H-31a7 81-62 GCACCCAAGACAGCAGAAA.G 55
H-3Ia8 81-62 GCACCCAAGACAGCAGAAAG 55
H-31a9 81-62 GCACCCAAGACAGCAGAAAG 55
H-3Ia10 81-62 GCACCCAAGACAGCAGAAAG 55
H-311 82-62 TGCACCCAAGACAGCAGAAAG 56
H-3I2 82-62 TGCACCCAAGACAGCAGAAAG 56
H-3I3 82-62 TGCACCCAAGACAGCAGAAAG 56
H-314 82-62 TGCACCCAAGACAGCAGAAAG 56
H-3I5 82-62 TGCACCCAAGACAGCAGAAAG 56
H-316 82-62 TGCACCCAAGACAGCAGAAAG 56
H-317 82-62 TGCACCCAAGACAGCAGAAAG 56
H-318 82-62 TGCACCCAAGACAGCAGAAAG 56
H-319 82-62 TGCACCCAAGACAGCAGAAAG 56
H-3110 82-62 TGCACCCAAGACAGCAGAAAG 56
H-3Jal 83-62 ATGCACCCAAGACAGCAGAAAG 57
H-3Ja2 83-62 ATGCACCCAAGACAGCAGAAAG 57
H-3Ja3 83-62 ATGCACCCAAGACAGCAGAAAG 57
H-3Ja4 83-62 ATGCACCCAAGACAGCAGAAAG 57
H-3Ja5 83-62 ATGCACCCAAGACAGCAGAAAG 57
H-3 Ja6 83-62 ATGCACCCAAGACAGCAGAAAG 57
H-3Ja7 83-62 ATGCACCCAAGACAGCAGAAAG 57
H-3Ja8 83-62 ATGCACCCAAGACAGCAGAAAG 57 H-3Ja9 83-62 ATGCACCCAAGACAGCAGAAAG 57
H-3Ja10 83-62 ATGCACCCAAGACAGCAGAAAG 57
H-3J1 84-62 AATGCACCCAAGACAGCAGAAAG 58
.H-3J2 84-62 AATGCACCCAAGACAGCAGAAAG 58
LT1 lTl w W N N H N
lJl 0 Ul 0 Ul 0 Ul 0 U7 0 U7
x i 0 O I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I
I I I I I I I I I 1 I I I I N N N W W W W W W W W W W U J W U J U J W U J W H
~
aoJ mcn~WNr~ICICk~CFCFCFCFCFC~C~C~C9C~C5C9C9C9C5C9C~c9c9C~C9C9C~C~Cb4y4~C1yq
~~ (7)
W N p O A1 A1 A1 A1 A1 W A1 A1 Q1 A1 F-' l0 OD ~l dl Ul rP W N h' Sll Sll QJ W
Sll W W A) W Ql P l0 00 < Ql Ul IA W 0 H'loOO -A OIUIA WNh'O h'loOO< OIInA WNF-
'O
0 0
~
H
wooaoaoao~aow~ooooaocu~waoo~oo~ooaowwooooaoo~m~ooaoaowwooaoaoaowaoaoooaoaoooooc
uwooooao U~~
l0 lD l0 00 0~ 0~ 00 00 00 0~ 00 00 0~ ~l ~l ~l ~l ~l ~l ~7 ~l ~l 01 61 Ql dl
61 dl Ol dl 61 Ol Ul Ul Ul U1 Ul Ul Ul Ul UI Ul FP EP A A FP FP IP ~P H~
I I I I 1 1 1 1 1 1 1 1 1 1 I 1 I I I I 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1
1 1 1 I I 1 1 1 1 1 1 1 Fj ~f '
mmmrnmmmrnmrnmmmmrnmmmmmmmmmmrnmmmmmmmmmmmmmmrnmm mmmmmmmarn trJH
N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N
N N N N N N N N N N N N CrJ
d
G~G1G~nnnnnnnnnnHHHHHHHHHHnnnnnnnnnnnnn nnnn(i
~~~~~~~~
nnnHHHHl3HH13HHnnnnnnnnnnnnnnnnnn ~~ y
HHHnnnnnnnnnnnnnnnnnnnn~~!~~~~'~~~~HHHHHHHHHHGHIGH~~GH~Gy]~~Q (n H N
nnn HHHHHyHyHHU~nu~u~nu~u~nu~c~nnnnn nn r~ N
q 1-3 q aaa a a a 0 a a a nnnnnnnnnn~~~~~~~~
~~~HHH~3HHHHHHG~G~G~G~G~G~G~G~G1Glnnnnnnnnn nnnnnnnn ~
yyy0 u) n0 (7) u) n(D 0 u)nnnnnnnnnnnnnnnnnnnnnnnnnnn t~ tv W
u~c~nnnnnnnnnnn~~~~~~~~~~nnnnnnnnnnnnnnnnnnnnnnnnnnnn z ~
n~n~~~~~~~~~~~~nnnnnnnnnnnnnnnnnnnnnnnnnnnnnn~~~~~~~~ n N ,
nnnnnnnnnnnnnnnnnnnnnnnnnnnnn~~~~~~~~~~
nnnnnnnnnnnnnn nn rr~rr LD
nnnnnnnnnnnnn0nnnu~c~c~nnn0
~~~~~~~ G~G~u~nu~u~G~nnu~P ~o,~,~vnnnnnnnn m 0
n
nnnnnnn
0 (7]G1G~ (7) rt
G~GIGI
nc~nonoooonnnnnnnn
nnnnnnnnnoc) c)u~nu~u~u~nnnnnnnnnnn~~
~> ~~u~u~u~nc~c~c~c~u~nnnnnnnnnn~~~~~~~ ~~~~u~Ou~u~u~u~
u~u~u~c~u~c~nu~u~c~~~~~~~~
nnnnnnnnnnnnn~~
nnn
Q a u)
rn
~
Ol Ol Ol O1 Ol Ol Ol Ol Ol Ol Ol O1 01 Ol Q1 Ol ol Ol dl Ql Ol Ol O1 Ol dl Ol
Ol Ol Ol Ol Ol 01 Ol U1 U1 U1 U1 U1 Ul U1 U1 Ul Ul U7 U1 Ul U1 U1 U1 U1 U1 ',Z
H U] O
W W W NNNNNNNNNNI'I f f'F'{F'I+I- I-
'OOOOOOOOOOIOIOIOIflIOIpIC~DlOl00D0~OD~0000pD00 QrJtrJ
1O A
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WO 96/27006 PCT/US96/02840
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TABLE 2 (aonti'.)
SEQ
TARGETED ID
OLIGO SITE SEOUENCE (5' --> 3') NO:
H-Za4 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 63
H-Za5 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 63
H-Za6 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 63
H-Za7 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 63
H-Za8 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 63
H-Za9 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 63
H-ZalO 89-62 GCTCCAATGCACCCAAGACAGCAGAAAG 63
H-Zl 90-62 GGCTCCAATGCACCCAAGACAGCAGAAAG 64
H-Z2 90-62 GGCTCCAATGCACCCAAGACAGCAGAAAG 64
H-Z3 90-62 GGCTCCAATGCACCCAAGACAGCAGAAAG 64
H-Z4 90-62 GGCTCCAATGCACCCAAGACAGCAGAAAG 64
H-Z5 90-62 GGCTCCAATGCACCCAAGACAGCAGAAAG 64
H-Z6 90-62 GGCTCCAATGCACCCAAGACAGCAGAAAG 64
H-Z7 90-62 GGCTCCAATGCACCCAAGACAGCAGAAAG 64
H-Z8 90-62 GGCTCCAATGCACCCAAGACAGCAGAAAG 64
H-Z9 90-62 GGCTCCAATGCACCCAAGACAGCAGAA.AG 64
H-Z10 90-62 GGCTCCAATGCACCCAAGACAGCAGAAAG 64
H-3D1 80-63 CACCCAAGACAGCAGAAA 65
H-3D2 80-63 CACCCAAGACAGCAGAAA 65
H-3D3 80-63 CACCCAAGACAGCAGAAA 65
H-3D4 80-63 CACCCAAGACAGCAGAA.A 65
H-3D5 80-63 CACCCAAGACAGCAGAAA 65
H-3D6 80-63 CACCCAAGACAGCAGAAA 65
H-3D7 80-63 CACCCAAGACAGCAGAAA 65
H-3D8 80-63 CACCCAAGACAGCAGAAA 65
H-3D9 80-63 CACCCAAGACAGCAGAAA 65
H-3D10 80-63 CACCCAAGACAGCAGAAA 65
H-3E1 80-64 CACCCAAGACAGCAGAA 66
H-3E2 80-64 CACCCAAGACAGCAGAA - 66
H-3E3 80-64 CACCCAAGACAGCAGAA 66
H-3E4 80-64 CACCCAAGACAGCAGAA 66
H-3E5 80-64 CACCCAAGACAGCAGAA 66
H-3E6 80-64 CACCCAAGACAGCAGAA 66
H-3E7 80-64 CACCCAAGACAGCAGAA 66 -
H-3E8 80-64 CACCCAAGACAGCAGAA 66
H-3E9 80-64 CACCCAAGACAGCAGAA 66
H-3E10 80-64 CACCCAAGACAGCAGAA 66
H-3F1 80-65 CACCCAAGACAGCAGA 67
H-3F2 80-65 CACCCAAGACAGCAGA 67
H-3F3 80-65 CACCCAAGACAGCAGA 67
H-3F4 80-65 CACCCAAGACAGCAGA 67
H-3F5 80-65 CACCCAAGACAGCAGA 67
H-3F6 80-65 CACCCAAGACAGCAGA 67
H-3F7 80-65 CACCCAAGACAGCAGA 67
H-3F8 80-65 CACCCAAGACAGCAGA 67
H-3F9 80-65 CACCCAAGACAGCAGA 67
H-3F10 80-65 CACCCAAGACAGCAGA 67
H-3G1 80-60 CACCCAAGACAGCAGAAAGTT 68
H-3G2 80-60 CACCCAAGACAGCAGAAAGTT 68
H-3G3 80-60 CACCCAAGACAGCAGAAAGTT 68
H-3G4 80-60 CACCCAAGACAGCAGAAAGTT 68
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TABLE
2 (conti'.)
SEQ
TARGETED ID
OLIGO SITE SEOUENCE (5' - 3') NO:
H-3G5 80-60 CACCCAAGACAGCAGAAAGTT 68
H-3G6 - 80-60 CACCCAAGACAGCAGAAAGTT 68
H-3G7 80-60 CACCCAAGACAGCAGAAAGTT 68
H-3G8 80-60 CACCCAAGACAGCAGAAAGTT 68
H-3G9 80-60 CACCCAAGACAGCAGAAAGTT 68
H-3G10 80-60 CACCCAAGACAGCAGAAAGTT 68
H-3H1 80-58 CACCCAAGACAGCAGAAAGTTCAT 69
H-3H2 80-58 CACCCAAGACAGCAGAAAGTTCAT 69
H-3H3 80-58 CACCCAAGACAGCAGAAAGTTCAT 69
H-3H4 80-58 CACCCAAGACAGCAGAAAGTTCAT 69
H-3H5 80-58 CACCCAAGACAGCAGAAAGTTCAT 69
H-3H6 80-58 CACCCAAGACAGCAGAAAGTTCAT 69
H-3H7 80-58 CACCCAAGACAGCAGAAAGTTCAT 69
H-3H8 80-58 CACCCAAGACAGCAGAAAGTTCAT 69
H-3H9 80-58 CACCCAAGACAGCAGAAAGTTCAT 69
H-3H10 80-58 CACCCAAGACAGCAGAAAGTTCAT 69
------------------------------------------------------------
Preferably, the nucleotides bolded in the oligonucleotides in
Table 2 above are 2'-0-alkylated, and all of the nucleotides are
linked via non-phosphodiester internucleotide linkages such as
phosphorothioates.
The preparation of these modified oligonucleotides is well
known - in the art (reviewed in Agrawal et al.(1992) Trends Biotechnol.
10 : 1 S 2-15 8); Agrawal et al.(199S) Curr. Opin. Biotechnol. 6:12 -19 ).
For example, nucleotides can be covalently linked using art-
recognized techniques such as phosphoramidate, H-phosphonate
chemistry, or methylphosphoramidate chemistry (see, e.g., Uhlmann et
al. (1990) Chen2. Rev. 90:543-584; Agrawal et al. (1987) Tetrahedron.
Lett. 28: (31) :3539-3542) ; Caruthers et al. (1987) Meth. Enzymol.
154:287-313; U.S. Patent 5,149,798). Oligomeric phosphorothioate
analogs can be prepared using methods well known in the field
such as methoxyphosphoramidite (see, e.g., Agrawal et al. (1988)
Proc. Natl. Acad. Sci. (USA) 85 : 7079-7083 ) or H-phosphonate (see, e.g.,
Froehler (1986) Tetrahedron Lett. 27:S575-5578) chemistry. The
synthetic methods described in Bergot et al. (J. Chromatog. (1992)
559:35-42) can also be used.
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The synthetic antisense oligonucleotides of the invention
in the form of a therapeutic formulation are useful in treating
diseases, and disorders, and conditions associated with
angiogenesis and neovascularization including, but not limited
to, retinal neovascularization, tumor growth, and wound healing.
In such methods, a therapeutic amount of a synthetic
oligonucleotide of the invention and effective in inhibiting the
expression of vascular endothelial growth factor is administered
to a cell. This cell may be part of a cell culture, a
neovascularized tissue culture, or may be part or the whole body
of an animal such as a human or other mammal. Administration may
be bolus, intermittent, or continuous, depending on the condition
and response, as determined by those with skill in the art. In
some preferred embodiments of the methods of the invention
described above, the oligonucleotide is administered locally
(e.g., intraocularly or interlesionally) and/or systemically.
The term "local administration" refers to delivery to a defined
area or region of the body, while the term "systemic
administration is meant to encompass delivery to the whole
organism by oral ingestion, or by intramuscular, intravenous,
subcutaneous, or intraperitoneal injection.
Such methods can be used to treat retinopathy of prematurity
(ROP), diabetic retinopathy, age-related macular degeneration,
sickle cell retinopathy, neovascular glaucoma, retinal vein
occlusion, and other hypoxic diseases.
The synthetic oligonucleotides of the invention may be used
as part of a pharmaceutical composition when combined with a
physiologically and/or pharmaceutically acceptable carrier. The
characteristics of the carrier will depend on the route of
administration. Such a composition may contain, in addition to
the synthetic oligonucleotide and carrier, diluents, fillers,
salts, buffers, stabilizers, solubilizers, and other materials
well known in the art. The pharmaceutical composition of the invention may
also contain other active factors and/or agents
which enhance inhibition of VEGF expression or which will reduce
CA 02214431 2005-02-28
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neovascularization. For example, combinations of synthetic
oligonucleotides, each of which is directed to different regions
of the VEGF mRNA, may be used in the pharmaceutical compositions
of the invention. The pharmaceutical composition of the
invention may further contain nucleotide analogs such as
azidothymidine, dideoxycytidine, dideosyinosine, and the like.
Such additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with
the synthetic oligonucleotide of the invention, or to minimize
side-effects caused by the synthetic oligonucleotide of the
invention. Conversely, the synthetic oligonucleotide of the
invention may be included in formulations of a particular anti-
VEGF or anti-neovascularization factor and/or agent to minimize
side effects of the anti-VEGF factor and/or agent.
The pharmaceutical composition of the invention may be in
the form of a liposome in which the synthetic oligonucleotides
of the invention is combined, in addition to other
pharmaceutically acceptable carriers, with amphipathic agents
such as lipids which exist in aggregated form as micelles,
insoluble monolayers, liquid crystals, or lamellar layers which
are in aqueous solution. Suitable lipids for liposomal
formulation include, without limitation, monoglycerides,
diglycerides, sulfatides, lysolecithin, phospholipids, saponin,
bile acids, and the like. One particularly useful lipid carrier
is lipofectin. Preparation of such liposomal formulations is
within the level of skill in the art, as disclosed, for example,
in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323. The
pharmaceutical composition of the invention may further include
compounds such as cyclodextrins and the like which enhance
delivery of oligonucleotides into cells,
or slow release polymers.
As used herein, the term "therapeutically effective amount"
means the total amount of each active component of the
pharmaceutical composition or method that.is sufficient to show
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a meaningful patient benefit, i.e., healing of chronic conditions
characterized by neovascularization or a reduction in
neovascularization, itself, or in an increase in rate of healing
of such conditions. When applied to an individual active
ingredient, administered alone, the term refers to that
ingredient alone. When applied to a combination, the term refers
to combined amounts of the active ingredients that result in the
therapeutic effect, whether administered in combination, serially
or simultaneously.
In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of one, two, or
more of the synthetic oligonucleotide oligonucleotides of the
invention is administered to a subject afflicted with a disease
or disorder related to neovascularization, or to a tissue which
has been neovascularized. The synthetic oligonucleotide of the
invention may be administered in accordance with the method of
the invention either alone of in combination with other known
therapies for neovascularization. When co-administered with one
or more other therapies, the synthetic oligonucleotide of the
invention may be administered either simultaneously with the
other treatment (s) , or sequentially. If administered
sequentially, the attending physician will decide on the
appropriate sequence of administering the synthetic
oligonucleotide of the invention in combination with the other
therapy.
Administration of the synthetic oligonucleotide of the
invention used in the pharmaceutical composition or to practice
the method of the present invention can be carried out in a
variety of conventional ways, such as intraocular, oral
ingestion, inhalation, or cutaneous, subcutaneous, intramuscular,
or intravenous injection.
When a therapeutically effective amount of synthetic
oligonucleotide of the invention is administered orally, the
synthetic oligonucleotide will be in the form of a tablet,
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capsule, powder, solution or elixir. When administered in tablet
form, the pharmaceutical composition of the invention may
additionally contain a solid carrier such as a gelatin or an
adjuvant. The tablet, capsule, and powder contain from about 5
to 95 o synthetic oligonucleotide and preferably from about 25 to
90% synthetic oligonucleotide. When administered in liquid form,
a liquid carrier such as water, petroleum, oils of animal or
plant origin such as peanut oil, mineral oil, soybean oil, sesame
oil, or synthetic oils may be added. The liquid form of the
pharmaceutical composition may further contain physiological
saline solution, dextrose or other saccharide solution, or
glycols such as ethylene glycol, propylene glycol or polyethylene
glycol. When administered in liquid form, the pharmaceutical
composition contains from about 0.5 to 90o by weight of the
synthetic oligonucleotide and preferably from about 1 to 500
synthetic oligonucleotide.
When a therapeutically effective amount of synthetic
oligonucleotide of the invention is administered by intravenous,
subcutaneous, intramuscular, intraocular, or intraperitoneal
injection, the synthetic oligonucleotide will be in the form of
a pyrogen-free, parenterally acceptable aqueous solution. The
preparation of such parenterally acceptable solutions, having due
regard to pH, isotonicity, stability, and the like, is within the
skill in the art. A preferred pharmaceutical composition for
intravenous, subcutaneous, intramuscular, intraperitoneal, or
intraocular injection should contain, in addition to the
synthetic oligonucleotide, an isotonic vehicle such as Sodium
Chloride Injection, Ringer's Injection, Dextrose Injection,.-
Dextrose and Sodium Chloride Injection, Lactated Ringer's
Injection, or other vehicle as known in the art. The
pharmaceutical composition of the present invention may also
contain stabilizers, preservatives, buffers, antioxidants, or
other additives known to those of skill in the art.
The amount of synthetic oligonucleotide in the
pharmaceutical composition of the present invention will depend
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upon the nature and severity of the condition being treated, and
on the nature of prior treatments which the patent has undergone.
Ultimately, the attending physician will decide the amount of
synthetic oligonucleotide with which to treat each individual
patient. Initially, the attending physician will administer low
doses of the synthetic oligonucleotide and observe the patient's
response. Larger doses of _synthetic oligonucleotide may be
administered until the optimal therapeutic effect is obtained for
the patient, and at that point the dosage is not increased
further. It is contemplated that the various pharmaceutical
compositions used to practice the method of the present invention
should contain about 10 g to about 20 mg of synthetic
oligonucleotide per kg body or organ weight.
The duration of intravenous therapy using the pharmaceutical
composition of the present invention will vary, depending on the
severity of the disease being treated and the condition and
potential idiosyncratic response of each individual patient.
Ultimately the attending physician will decide on the appropriate
duration of intravenous therapy using the pharmaceutical
composition-of the present invention.
Some diseases lend themselves to acute treatment while
others require to longer term therapy. Proliferative retinopathy
can reach a threshold in a matter of days as seen in ROP, some
cases of diabetic retinopathy, and neovascular glaucoma.
Premature infants are at risk for neovascularization around what
would be 35 weeks gestation, a few weeks after birth, and will
remain at risk for a short period of time until the retina
becomes vascularized. Diabetic retinopathy can be acute but may
also smolder in the proliferative phase for considerably longer.
Diabetic retinopathy will eventually become quiescent as the
vasoproliferative signal diminishes with neovascularization or
destruction of the retina.
Both acute and long term intervention in retinal disease are
worthy goals. Intravitreal injections of oligonucleotides
CA 02214431 2005-02-28
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against VEGF can be an effective means of inhibiting retinal
neovascularization in an acute situation. However for long term
therapy over a period of years, systemic delivery
(intraperitoneal, intramuscular, subcutaneous, intravenous)
either with carriers such as saline, slow release polymers, or
liposomes should be considered.
In some cases of chronic neovascular disease, systemic
administration of oligonucleotides may be preferable. Since the
disease process concerns vessels which are abnormal and leaky,
the problem of passage through the blood brain barrier may not
be a problem. Therefore, systemic delivery may prove
efficacious. The frequency of injections is from continuous
infusion to once a month, depending on the disease process and
the biological half life of the oligonucleotides.
In addition to inhibiting neovascularization in vivo,
antisense oligonucleotides specific for VEGF are useful in
determining the role of this cytokine in processes where
neovascularization is involved. For example, this technology is
useful in in vitro systems which mimic blood vessel formation and
permeability, and in invivo system models of neovascularization,
such as the murine model described below.
A murine model of oxygen-induced retinal neovascularization
has been established which occurs in 1000 of treated animals and
is quantifiable (Smith et al. (1994) Invest. Ophthalmol. Vis. Sci. 35:101-
111). Using this model, a correlation has been determined
between increasing expression of VEGF message and the onset of
retinal neovascularization in the inner nuclear and ganglion cell
layers ( i. e., in Miiller cells).
This result has been confirmed by
Northern blot and in situ hybridization analysis of whole retinas
at different' time points during the development of
neovascularization (Pierce et al., ibid.).
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Oligonucleotides of the invention are also useful in a
method of reducing the expression of VEGF. The target VEGF
expression can be in vitro or in any cell which expresses VEGF.
In this method, nucleic acid specific for VEGF is contacted with
at oligonucleotide of the invention such that transcription of
the nucleic acid to mRNA and/or protein is reduced or inhibited.
That oligonucleotides of the invention can inhibit VEGF
expression at the protein level can be demonstrated using an
ELISA which specifically detects human VEGF and a VEGF-expressing
cell line such as a human glioblastoma (e.g., U373 ATCC Ac. no.
HTB17, American Type Culture Collection, Rockville, MD) or a
human melanoma (e.g.,SK-MEL-2, ATCC Ac. no. HTB68, American Type
Culture Collection, Rockville, MD; or M21). Briefly, when a
human glioblastoma cell line U373 and a human melanoma cell line
M21 were treated with VEGF-specific oligonucleotides of the
invention, these cells stop expressing VEGF in a sequence-
specific manner, as shown in FIGS. 7, 8, 9, and 10, respectively.
FIG. 11 demonstrates that modification of the oligonucleotides
does not reduce their inhibitory activity. Oligonucleotides of
the invention also reduced VEGF mRNA expression, as demonstrated
by the Northern analyses described in EXAMPLE 4 below.
The role of VEGF in tumor formation in vivo can be
demonstrated using an athymic mouse injected with as an animal
model. M21 cells are known to generate palpable tumors in mice
in about 1 to 1.5 weeks. Alternately, a U373 cell line which has
been passed through an athymic mouse in the presence of
Engelbreth Holm Swarm (EMS) tumor matrix (MatrigelTm,
Collaborative Research, Waltham, MA) may be used. When mice are
injected with VEGF-specific oligonucleotides of the invention,
there will be a reduction in tumor weight and volume if VEGF
expression is reduced by oligonucleotides or pharmaceutical
formulations of the invention.
That VEGF plays a role in retinal neovascularization has
been shown using the murine model of neovascularization described
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above. Three independent 'experiments were performed using
antisense oligonucleotides specific for VEGF (JG-3 (SEQ ID NO:
47), JG-4, (SEQ ID NO: 48), and Vm (SEQ ID NO: 46), and a
corresponding sense oligonucleotide (V2 (SEQ ID NO: 49). These
oligonucleotides were designed using the known nucleic sequence
of murine VEGF (Claffee et al. (1992) J. Biol. Chem. 267:16317-
16322). The sequence of the Vm oligonucleotide (SEQ ID NO:46)
is targeted to the sequence surrounding the translational TGA
stop site (TGA). The sequence of JG-4 (SEQ ID NO:48) is targeted
to the sequence 5' to and containing the ATG of the translational
start site of the murine VEGF molecule. The sequence of JG-3
(SEQ ID NO:47) is targeted to the 5' untranslated region, and the
V2 sense sequence is targeted to the sequence surrounding the
translational start site (ATG). A compilation of the results of
these experiments is presented in FIG. 2. These results indicate
that Vm (SEQ ID NO:46) antisense oligonucleotide significantly
reduces retinal neovascularization when compared with both
untreated and sense oligonucleotide V2, (SEQ ID NO:49.) controls.
JG-3 (SEQ ID NO:47) and JG-4 (SEQ ID NO:47) show significant
activity when compared against untreated eyes. The sense control
oligonucleotide V2 (SEQ ID NO:49) does not show any significant
activity when compared with untreated eyes.
In the studies described above, the human VEGF antisense
oligonucleotide which corresponds to murine JG-3 is H-1 (SEQ ID
NO:l), which is targeted to the 5' untranslated region; that
which corresponds to murine JG-4 is H-17 (SEQ ID NO:30), which
is targeted to the sequence 5' to and containing the ATG of the
translational start site of the human VEGF molecule; and that
which corresponds to the murine Vm gene is VH (SEQ ID NO : 4 6),
which is targeted to sequences surrounding the translational stop
site (TGA) of the human VEGF molecule. These antisense
oligonucleotides of the invention are expected to inhibit VEGF
expression in human cells in much the same way as the murine
antisense oligonucleotides inhibit expression of VEGF in mouse
cells.
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Human VEGF antisense sequences corresponding to other murine
sequences are also known. For example, human oligonucleotide H-6
(SEQ ID NO:12) corresponds to a region spanning murine sequences
JG-6 (SEQ ID NO:52) and JG-7 (SEQ ID NO:53), and human
oligonucleotide H-2 (SEQ ID NO:5) is in the same region as murine
sequence JG-5 (SEQ ID NO:51). It is likely that these sequences
have a similar effect on inhibition of VEGF expression and hence
on controlling neovascularization.
There are several methods by which the effects of antisense
oligonucleotides on VEGF expression and neovascularization can
be monitored. One way is a capture ELISA developed for
quantifying human VEGF protein expressed by cells. Using this
assay, it has been determined that an antisense phosphorothioate
oligonucleotide H-3 (SEQ ID NO:6) targeted to a sequence just 3'
to the translational start site can inhibit the hypoxic induction
of VEGF expression in a sequence-specific manner, compared with
random (R) and sense (H-16, SEQ ID NO:29) controls), as shown in
FIG. 4. This inhibition is reproducible and in this in vitro
system appears to be lipid carrier-specific and antisense-
specific as only antisense oligonucleotide H-3 (SEQ ID NO:6) in
the presence of lipofectin (a lipid carrier), and not
lipofectamine (another lipid carrier),
results in inhibition of VEGF protein expression.
At the RNA level, Northern blots (Sambrook et al. (1989)
Molecular Cloning; a Laboratory Manual, Cold Spring Harbor Laboratory
Press, NY, Vol. 1, pp. 7.38; Arcellana-Panlilio et al. (1993)
Meth. Enz. 225:303-328) can be performed to determine the extent
that oligonucleotides of the invention inhibit the expression of
VEGF mRNA. For example, as shown in FIG. 5, a histogram
representing Northern blot analysis demonstrates a decrease in
VEGF RNA levels in culture human cells treated with antisense
oligonucleotide H-3 (SEQ ID NO:6), while there is only a minimal
change in VEGF RNA levels in samples treated with sense control
H-16 (SEQ ID NO:29).
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In addition, bioactivity can be determined by several
methods, including the Miles vessel permeability assay (Miles and
Miles (1952) J. Physiol. (Lond. ) 118:228), which measures vessel
permeability, endothelial cell mitogenicity, which measures cell
growth, and intracellular calcium release in endothelial cells
(see, e.g. , Brock and Capasso (1988) J. Cell. Physiol. 136:54) , which
measures the release of calcium in response to VEGF binding to
its receptor on endothelial cells.
The following examples illustrate the preferred modes of
making and practicing the present invention, but are not meant
to limit the scope of the invention since alternative methods may
be utilized to obtain similar results.
EXAMPLE 1
PREPARATION OF VEGF-SPECIFIC OLIGONUCLEOTIDES
Human VEGF cDNA is transcribed in vitro using an in vitro
eukaryotic transcription kit (Stratagene, La Jolla, CA). The RNA
is labelled with 32P using T-4 polynucleotide kinase as described
by (Sambrook et al. (1989) Molecular Cloning; a Laboratory Manual, Cold
Spring Harbor Laboratory Press, NY, Vol. l, pp. 5.71). The
labelledRNA is incubated in the presence of a randomer 20mer
library and RNAse H, an enzyme which cleaves RNA-DNA duplexes
(Boehringer Mannheim, Indianapolis, IN). Cleavage patterns are
analyzed on a Go polyacrylamide urea gel. The specific location
of the cleaved fragments is determined using a human VEGF
sequence ladder (Sequenase Kit, United States Biochemical,
Cleveland, OH).
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EXAMPLE 2
ANIMAL MODEL OF RETINAL NEOVASCULARIZATION
A. Preparation of Oligonucleotides
Synthesis of the following oligonucleotides: JG-3 (SEQ ID
NO:47), JG-4 (SEQ ID NO:48), Vm (SEQ ID NO:46), and V2 (SEQ ID
NO:49), was performed on a PharmaciaM Gene Assembler series
synthesizer using the phosphoramidite procedure (see, e.g.,
Uhlmann et al. (Chem.Rev. (1990) 90:534-583). Following assembly
and deprotection, oligonucleotides were ethanol precipitated
twice, dried, and suspended in phosphate-buffered saline (PBS)
at the desired concentration.
The purity of these oligonucleotides was tested by capillary
gel electrophoreses and ion exchange HPLC. Endotoxin levels in
the oligonucleotide preparation wascietermined using the Luminous
Amebocyte Assay (Bang (1953) Biol. Bull. (Woods Hole, MA) 105:361-
362).
B. Preparation of Animal Model
Seven day postnatal mice (P7, C57b1/6J, (Children's Hospital
Breeding Facilities, Boston, MA) were exposed to 5 days of
hyperoxic conditions (75 +/- 2%) oxygen in a sealed incubator
connected to a Bird 3-M oxygen blender (flow rate: 1.5
liters/minute; Bird, Palm Springs, CA) . The oxygen concentration
was monitored by means of an oxygen analyzer (Beckman Model D2,
Irvine, CA) . After 5 davs (P12), the mic-e were returned to room
air. Maximal retinal neovascularization was observed 5 days
after return to room air (P17) . After P21, the level of retinal
neovascularization was just beginning to regress.
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C. Treatment
After mice had been removed from oxygen, antisense
oligonucleotides were injected into the vitreous with a Hamilton
syringe and a 33 gauge needle (Hamilton Company, Reno, NV). The
animals were anesthetized for the procedure with Avertin ip. The
mice were given a single injection of antisense oligonucleotides
(or sense or non-sense controls) at P12 achieving a final
concentration of approximately 30 M. The animals were
sacrificed at P17 with tribromoethanol ip (0.1 ml/g body weight)
and cervical dislocation.
D. Microscopy
The eyes were enucleated, fixed in 4% paraformaldehyde, and
embedded in paraf f in . Serial sections of the whole eyes were cut
sagittally, through the cornea, and parallel to the optic nerve.
The sections were stained with hematoxylin and periodic acid-
Schiff (PAS) stain. The extent of neovascularization in the
treated eyes was determined by counting endothelial cell nuclei
extending past the internal limiting membrane into the vitreous.
Nuclei from new vessels and vessel profiles could be
distinguished from other structures in the retina and counted in
cross-section with light microscopy. Additional eyes were
sectioned and examined by insitu hybridization to a VEGF probe.
To examine the retinal vasculature using fluorescein-
dextran, the mice were perfused with a 50 mg/mi solution of high
molecular weight fluorescein-dextran (Sigma Chemical Company, St.
Louis, MO) in 4% paraformaldehyde. The eyes were enucleated,
fixed in paraformaldehyde, and flat-mounted with glycerol-
gelatin. The flat-mounted retinas were viewed and photographed
by fluorescence microscopy using an OlympusMBX60 fluorescence
rnicroscope (OlympusMAmerica Corp., Bellingham, MA).
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EXAMPLE 3
RETINOPATHY OF PREMATURITY
A. Preparation of Oligonucleotides
VEGF specific oligonucleotides are synthesized as described
in EXAMPLE 2A above. Sterile and endotoxin-free oligonucleotides
are diluted in Balanced Salt Solution (BSS, Alcon, Fort Worth,
TX) so as to have the same pH and electrolyte concentration as
the aqueous or vitreous of the eye. Emalphor EC620 (2.5a, GAF
Corp.) (Bursell et al. (1993) J. Clin. Invest. 92:2872-2876) , a
petroleum product, is added to change viscosity and aid in
delivery properties. Doses to achieve intravitreal
concentrations ranging from 0.1 M - 100 M are administered
depending on the severity of the retinal/ocular
neovascularization. The volume delivered is between 1 l and 1
ml depending on the volume of the eye.
B. ROP Patient Profile
The patient treated is a premature, 34 week post-conception
Caucasian female weighing less than 1,000 grams at birth and is
respirator-dependent. The patient has bilateral stage 3+ disease
with 11 clock hours of neovascularization in each eye. There is
hemorrhaging in one eye, and both eyes have reached "threshold"
according to the international classification (i.e., each eye has
>50a chance of going on to retinal detachment) . Extraretinal
fibrovascular proliferation is found in both eyes.
C. Treatment
The intubated patient is anesthetized with fluorane. The
face and eyes are prepared with a betadine scrub and draped in
the usual sterile fashion. The sterile drug with vehicle is
injected with a 33 gauge needle on a sterile syringe at the
posterior limbus (pars plana) through full thickness sclera into
the vitreous. No closing suture is required unless there is
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leakage. Antibiotic drops containing gentamicin or erythromycin
ointment is applied to the surface of the globe in the palpebral
fissure several times per day until there is complete wound
closure. The frequency of injection ranges from every other day
to once every 6 months or less, depending on the severity of the
disease process, the degree of intraocular inflammation, the
character of the vehicle (i.e., slow release characteristics),
the degree of inhibition of the neovascularization and the
tolerance of the eye to injections. Short and long term follow-
up check-ups for possible retinal detachment from the neovascular
disease as well as from the injections are necessary.
D. Monitoring of Progress
The eye upon dilation is monitored for signs of
inflammation, infection, and resolution of neovascularization by
both a direct and a indirect ophthalmoscope to view the retina
and fundus. A slit lamp exam is used in some cases of anterior
segment disease. Positive response to treatment includes fewer
neovascular tufts, fewer clock hours of involvement, and less
tortuosity of large blood vessels. Monitoring can be as frequent
as every day in cases where premature infants are threatened with
retinal detachment from proliferative ROP. The frequency of
monitoring will diminish with resolution of neovascularization.
EXAMPLE 4
DIABETIC RETINOPATHY
A. Preparation of Oligonucleotides
VEGF specific oligonucleotides are synthesized as described
in EXAMPLE 2A above and prepared for administration as described
in EXAMPLE 3A above. Doses to achieve intravitreal
concentrations ranging from 0.1 - 100 AM are administered
depending on the severity of the retinal/ocular
neovascularization. The volume delivered is between 1 l and 1
ml depending on the volume of the eye and whether vitreous has
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been previously removed as during a vitrectomy for diabetic eye
disease.
B. Diabetic Patient Profile
The patient to be treated is a 30 year old African American
male suffering for 25 years from juvenile-onset diabetes. The
patient has bilateral proliferative retinopathy with sub-retinal
hemorrhaging, cotton wool spots, and exudates. Upon fluorescein
angiography, there are well defined areas of neovascularization
bilaterally with areas of capillary drop-out.
C. Treatment
The patient is treated weekly with intraocular injections
of oligonucleotides resuspended in the appropriate vehicle (BSS,
Emanfour) at concentrations within the range of 0.1 - 100 M.
The treatment may be supplemented with systemic delivery of
oligonucleotide (i.e., intravenous, subcutaneous, or
intramuscular) from 2 to 5 times per day to once a month,
depending on the disease process and the biological half life of
the oligonucleotides.
D. Monitoring of Progress
The patient's eyes are monitored as described above in
EXAMPLE 2D. The eyes upon dilation are examined for regression
of neovascularization with both a direct and an indirect
ophthalmoscope to view the retina and fundus. A slit lamp exam
is used in the case of anterior segment disease. Repeat
injections are given as needed, based on the degree of inhibition
of the neovascularization and the tolerance of the eye to
injections. Short and long term follow-up check-ups are given
to check for possible retinal detachment from the neovascular
disease as well as from the injections.
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EXAMPLE 5
AGE-RELATED MACULAR DEGENERATION
A. Preparation of Oligonucleotides
VEGF specific oligonucleotides are synthesized as described
in EXAMPLE 2A above and prepared as described in EXAMPLE 3A
above. Doses to achieve intravitreal concentrations ranging from
1 l and 1 ml are administered depending on the severity of the
retinal/ocular neovascularization. The volume delivered is
between 1 l and 1 ml depending on the volume of the eye and
whether vitreous has been previously removed.
B. ARMD Patient Profile
The patient is a 50 year old Caucasian male suffering from
the exudative form of age related macular degeneration. This
patient has choroidal neovascularization which is apparent from
fluorescein angiography. The disease is bilateral and the
patient has a reduction in vision in each eye from 20/60 to
20/100.
C. Treatment
The patient is treated weekly with intraocular injections
of oligonucleotide resuspended in the appropriate vehicle (BSS,
Emanfour) at concentrations within the range of 0.1 to 100 M.
This treatment may be supplemented with systemic delivery of-
oligonucleotide (i.e., . intravenous, subcutaneous, or
intramuscular) from 2 to 5 times per day to once a month.
D. Monitoring of Progress
The eyes upon dilation are examined for regression of
neovascularization with both a direct and an indirect
ophthalmoscope to view the retina and fundus. Fluorescein
angiography is used to check for the resolution of
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neovascularization. A slit lamp exam is used in the case of
anterior segment disease. Repeat injections are given as needed,
based on the degree of inhibition of the neovascularization and
the tolerance of the eye to injections. Short and long term =
follow-up check-ups are given to check for possible retinal
detachment from the neovascular disease as well as from the
injections.
EXAMPLE 6
HUMAN CELL CULTURE
U373 human neuroblastoma cells were cultured in Dulbecco's
modified Earls (DME) medium containing glucose (4500 mg/ml) and
glutamate (2 mM) (Mediatech, Washington, DC) supplemented-with
penicillin/streptomycin (100 IU/MI/100 mcg/ml, Mediatech,
Washington, DC). The cells were cultured at 37 C under 10% CO2.
The cells were plated in 96 well tissue culture dishes (Costar
Corp., Cambridge, MA) and maintained as above. The cells were
placed under anoxic conditions for 18-20 hours using an anaerobic
chamber (BBL Gas Pak, Cockeysville, MD) or in the presence of 250
M CoC12 .
EXAMPLE 7
NORTHERN BLOTTING
In order to determine the level at which inhibition of VEGF
expression occurs in cells in the presence of an oligonucleotide
of the invention, Northern blotting was carried out. Human U373
cells cultured as described in EXAMPLE 6 above were plated in 100
mm tissue culture dishes and treated for 12 hours in the presence
of 5 g/ml lipofectin (Gibco-BRL, Gaithersburg, MD) as a lipid
carrier with oligonucleotide H-3 (SEQ ID NO:6) (antisense
oligonucleotide) and H-16 (SEQ ID NO:29) (sense oligonucleotide)
at 0.05 M, 0.5 M, and 2.0 M, respectively. The cells were
refed after 12' to 15 hours with fresh media + oligonucleotide
(minus lipofectin) and allowed to recover for 5 to 7 hours. The
cells were placed in hypoxia for 18 to 20 hours total RNA was
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isolated using the single-step acid guanidinium thiocyanate-
phenol-chloroform extraction method described by Chomczynski and
Sacchi (Anal. Biochem. (1987) 162:156-159) . Northern blotting was
performed according to the methods of Sambrook et al. (Molecular
Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, NY)
(1989) Vol. 1, pp. 7.38) or Arcellana-Panlilio et al. (Meth. Enz.
(1993) 225:303-328) All RNA signals were quantified on a
Phosphorimager (BioRad, Hercules, CA) and normalized using the
3 6B4 cDNA probe (Laborda (1991) Nucleic Acids Res. 19 : 3 9 9 8). RNA
expression was reduced in the presence of VEGF-specific
oligonucleotides of the invention, and is not significantly
affected by the presence of control sense oligonucleotide.
EXAMPLE 8
ELISA VEGF PROTEIN STUDY
U373 neuroblastoma cells as described in EXAMPLE 6 above
were plated in a 96 well tissue culture dish and treated
overnight with varying concentrations of antisense
oligonucleotides against human VEGF in the presence of 5 g/ml
lipofectin. The cells were refed after 12 to 15 hours with fresh
media + oligonucleotide (no lipofectin) and allowed to recover
for 5 to 7 hours. The dishes were placed under hypoxic
conditions for 18 to 20 hours using an anaerobic chamber (Gas
Pac, Cockeysville, MD). The media was analyzed using the antigen
capture ELISA assay described above (approximately 36 hours post
treatment). The human VEGF oligonucleotides used were H-3 (SEQ
ID NO:6) (antisense, coding), H-16 (SEQ ID NO:29) (start
site/coding, sense control), and a random control (R).
The culture medium from the cells described in EXAMPLE 5 was
analyzed for VEGF protein as follows. 96-well plates (Maxizorb
ELISA Nunc A/S, Camstrup, Denmark) were treated overnight at 4 C
with 100 l/well of the capture antibody, a monocional antibody
against human VEGF (R&D Systems, Minneapolis, MN, 2.5 /.cg/ml in
ix PBS). The wells were washed three times with 1X PBS/0.050
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TM
Tween-20 (United States Biochemical, Cleveland, OH) using a plate
washer (Dynatech, Gurnsey Channel Islands) . Non-specific binding
sites in the wells were blocked by adding 2k normal human serum
(100 l) and incubating the plate at 37 C for 2 hours. This
blocking solution was removed and 200 l conditioned medium
containing human VEGF added to each well and incubated at 37 C
for 2 to 3 hours. The plates were washed as described above.
100 l of the primary antibody (618/619, 2 g/ml in normal human
serum) was added to each well and incubated at 37 C for 1 to 2
hours. The secondary antibody was an affinity purified rabbit
anti- human VEGF polyclonal) . The plates were washed as
described above. 100 l of the detection antibody, a horse
radish peroxidase-labelled goat anti-mouse IgG monoclonal
antibody (1:10,000, Vector Laboratories, Burlinggame, CA), was
added to each well and incubated at 37 C for 1 hour. The plates
were washed as described above. The wells were developed using
the TMB microwell peroxidase developing system (Kirkegaard and
Perry, Gaithersburg, MD), and quantified at 450 nm using a Ceres
900 plate reader (Bio-Tek Instruments, Inc., Winooski, Vermont).
The linear range of this assay is between 2 ng and 0.01 ng human
VEGF. Representative results are shown in FIG. 3.
EXAMPLE 9
BIOACTIVITY ASSAYS
Bioactivity can be determined by the Miles vessel
permeability assay (Miles and Miles (1952) J. Physiol. (Lond.)
118:228). Briefly, Hartley guinea pigs (800 g) are shaved and
depilated and injected intravenously with 1.0 ml of normal saline
containing 0.5 g of Evans Blue dye per 100 ml. Subcutaneous
injections (250 l) of serum-free medium containing unknown
quantities of VEGF are performed. Positive (purified VEGF) and
negative controls (normal saline) are also included in the
experiment. Twenty minutes post-injection, the animals are
sacrificed and the test and control sites are cut out and
quantitated for extravasation of Evans Blue dye. The limit of
detection for this assay is 500 pM.
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Endothelial cell mitogenicity can also manifest bioactivity.
In this method, human umbilical vein endothelial (HUVEC) are
grown and maintained using the Biocoat endothelial cell growth
environment (Collaborative Biomedical Products, Bedford, MA).
1 x 10 cells are then plated in duplicate on 35 mM tissue
culture dishes in 1.4 ml E-STIM medium (Collaborative Biomedical
Products, Bedford, MA) plus 5%- heat-inactivated fetal bovine
serum. Following cell attachment (about 4 hours), two dishes of
cells are trypsinized, counted, and used for a starting cell
number. Test samples containing unknown amounts of VEGF are then
added in duplicate to the remaining dishes at day 0 and at day
2. Controls consisting of purified VEGF (positive) and PBS
(negative) are also used. On day 4, the dishes of cells are
trypsinized, counted and compared to the starting cell number.
The limit of detection for this assay is 10 pM.
The intracellular calcium release assay is also used to
monitor bioactivity (see, e.g., Brock and Capasso (1988) J. Cell.
Physiol. 136:54) . Human umbilical vein endothelial cells (HUVEC)
are maintained in EGM-UV medium. Cells are removed from the
plate by means of EDTA and collagenase. The calcium-sensitive
dye, Fura-2 (Molecular Probes, Eugene, OR), is used to monitor
changes in the concentration of intracellular calcium. In brief,
medium containing an unknown concentration of VEGF is added to
an aliquot of suspended HUVEC, pre-loaded with Fura-2. Changes
in fluorescence representing changes in intracellular calcium
release are measured using a HitachiTm 2000 F fluorometer.
Positive (histamine, thrombin) and negative (EGTA) controls are
also analyzed. This method is extremely sensitive and has a
limit of detection of 0.2 pM.
EXAMPLE 10
ELISA VEGF PROTEIN STUDY
U373 glioblastoma cells were plated in a 96 well tissue
culture dish and treated overnight with varying concentrations
of antisense oligonucleotides against human VEGF in the presence
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of 5 g/ml lipofectin. The cells were refed after 7 to 12 hours
with fresh media and allowed to recover for 5 to 7 hours. The
dishes were placed under hypoxic conditions for 18 to 20 hours
using an anaerobic chamber (Gas Pac, Cockeysville, MD) or in the
presence of 250 M CoC1Z. Cells maintained under normoxic
conditions served as uninduced controls. The media was analyzed
using the antigen capture ELISA assay described below
(approximately 24 hours post treatment).
The culture medium from the cells described in EXAMPLE 2 was
analyzed for VEGF protein as follows. 96-well plates (Maxizorb
ELISA Nunc A/S, Camstrup, Denmark) were treated overnight at 4 C
with 1o0 l/well of the capture antibody, a monoclonal antibody
against human VEGF (R&D Systems, Minneapolis, MN, 2.5 g/ml in
1X PBS). The wells were washed three times with lx PBS/0.05%
M
T~ween-20T(United States Biochemical, Cleveland, OH) using a plate
washer (DynaI tech, Gurnsey Channel Islands). Non-specific binding
sites in the wells were blocked by adding 2% normal human serum
(200 l) and incubating the plate at 37 C for 2 hours. This
blocking solution was removed and 200 l conditioned medium
containing human VEGF added to each well and incubated at 37 C
for 2 to 3 hours or overnight at 4 C. The plates were washed as
described above. 100 l of the primary antibody (618/619, 2
g/m1 in normal human serum) was added to each well and incubated
at 37 C for 1 to 2 hours. The primary antibody was an affinity
purified rabbit anti-human VEGF polyclonal)= The plates were
washed as described above. 100 l of the detection antibody, a
horse radish peroxidase-labelled goat anti-rabbit IgG monoclonal
antibody (1:10,000, Vector Laboratories, Burlinggame, CA), was
added to each well and incubated at 37 C for 1 hour. The plates
were washed as described above. The wells were developed using
the TMB microwell peroxidase developing system (Kirkegaard and
Perry, Gaithersburg, MD), and quantified at 450 nm using a Ceres
900 plate reader (Bio-Tek Instruments, Inc., Winooski, Vermont).
The linear range of this assay is between 2 ng and 0.01 ng human
VEGF. Representative results are shown in FIGS. 2-6.
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EXAMPLE 11
IN VIVO STUDIES
TM
A. Matrigel Studies
U373 glioblastoma cells were treated with 0.5 M antisense
phosphorothioate oligodeoxynucleotide (H-3, SEQ ID NO:54) or
control (H3-sense phosphorothioate oligonucleotide; SEQ ID N0:74)
for 7 hours in the presence of 5 g/ l Lipofectin (Gibco-BRL,
Gaithersburg, MD). 1 x 106 oligonucleotide-treated cells were
mixed with 250 l Matrigel' (Collaborative Research, Waltham,
MA; 10-12 mg/ml) and injected subcutaneously into 6-8 week old
athymic mice (about 20 g) (Charles River Laboratories,
Wilmington, MA) on both the left and right sides. These cells
respond to hypoxia and express increased levels of VEGF. The
mice were maintained ad libitum and sacrificed 8 days post
TM
injection. The skin was dissected to expose the Matrigel pellet.
Gross photography of the surrounding blood vessels was performed
with a ZeissMMacroscope. The Matrigel TM plugs were removed and
fixed in formalin for paraffin embedding and histological
analysis. Tissue sections were stained with hematoxylin and
TM
eosin for quantitation of blood vessel growth into the Matrigel
plug.
TM
The injection of Matrigel alone resulted in a clear plug
TM
with no apparent angiogenesis. Matrigel plugs combined with U373
glioblastoma cells contained visible hemhorraging. In addition,
the capillary bed surrounding the plug was more dense and the
blood vessels were more tortuous. Athymic mice injected with
MatrigelMplugs combined with antisense oligonucleotide-treated
cells generated less angiogenesis than the mice injected with
TM
Matrigel plugs and either untreated cells or cells pretreated
with the control oligonucleotide. MatrigelTMplugs containing
antisense treated cells also had less visible hemhorraging. The
results suggest that antisense oligonucleotide treatment inhibit
VEGF-induced angiogenesis.
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B. Tumor Studies
6 week old athymic mice (about 20 g) are purchased from
Charles River Laboratories. Human melanoma M21 cells or human
glioblastoma U373 cells which have been passaged through athymic
mice in the presence of Matrigel are injected subcutaneously (2-
20 x 106) into the flank of athymic mice. Palpable tumors are
generated in 1-2 weeks. Subcutaneous antisense or sense control
oligonucleotide injections begin one day following the injection
of the tumor cells. The concentration of oligonucleotide is
determined and ranges between 5 and 50 mg/kg. Animals are then
injected over a period of three weeks. They are then sacrificed
and the tumors removed. Tumors are analyzed initially for weight
and volume. In addition, analysis includes sectioning and
staining for VEGF/VPF protein using an anti- human VEGF/VPF
monoclonal antibody (R&D Systems, Minneapolis, MN) or VEGF/VPF
RNA using in situ hybridization techniques. Mice injected with
antisense oligonucleotides of the invention are expected to have
smaller tumors than those injected with vehicle or the control.
EQUIVALENTS
Those skilled in the art will recognize, or be able to
ascertain, using no more than routine experimentation, numerous
equivalents to the specific substances and procedures described
herein. Such equivalents are considered to be within the scope.
of this invention, and are covered by the following claims.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Hybridon, Inc.
(ii) TITLE OF INVENTION: Human VEGF-Specific
Oligonucleotides
(iii) NUMBER OF SEQUENCES: 74
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Lappin & Kusmer
(B) STREET: 200 State Street
(C) CITY: Boston
(D) STATE: Massachusetts
(E) COUNTRY: USA
(F) ZIP: 02109
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE:
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Kerner, Ann-Louise
(B) REGISTRATION NUMBER: 33,523
(C) REFERENCE/DOCKET NUMBER: HYZ-031CPPCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617-330-1300
(B) TELEFAX: 617-330-1311
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i i) MOLECULE TYPE : cDNA
= (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
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CGCCGGGCCG CCAGCACACT 20
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CGCCGGGCCG CCAGCACACU 20
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GGCCGCCAGC ACACT 15
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
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GCTCGCGCCG GGCCGCCAGC ACACT -25
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CAAGACAGCA GAAAGTTCAT 20
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid -- -
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CACCCAAGAC AGCAGAAAG 19
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid - -
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
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CACCCAAGAC AGCAG 15
(2) INFORMA.TION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPQLOGY: linear
(ii) MOLECULE TYPE: ODNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CCAATGCACC CAAGACAGCA GAAAG 25
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ACGCACACAG AACAAGACG 19.
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
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AAGTTCATGG TTTCGGAGGC 20
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GTTCATGGTT TCGGAGGCCC 20
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GTGCAGCCTG GGACCACTTG 20
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
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-50-
CGCCTCGGCT TGTCACATCT 20
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14c
CTTCCTCCTG CCCGGCTCAC 20
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CUUCCUCCUG CCCGGCUCAC 20
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(.C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1_6:
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-51-
CTTCCTCCTG CCCGG 15
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2S base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GGCTCCTTCC TCCTGCCCGG CTCAC 25
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GTCTCCTCTT CCTTCATTTC 20
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
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-52-
GTCTCCTCTT CCTTC 15
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GCAGAGTCTC CTCTTCCTTC ATTTC 25
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CGGACCCAAA GTGCTCTGCG 20
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
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-53-
CCAAAGTGCT CTGCG 15
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CCCTCCGGAC CCAAAGTGCT CTGCG - 25
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GGGCACGACC GCTTACCTTG 20
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
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-54-
GGGACCACTG AGGACAGAAA 20
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
CACCACTGCA TGAGAGGCGA 20
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
TCCCAAAGAT GCCCACCTGC 20
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
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-55-
CGCATAATCT GGAAAGGAAG 20
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
ACTTTCTGCT GTCTTGGGTG 20
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
CATGGTTTCG GAGGCCCGAC 20
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
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-56-
CAUGGTTUCG GAGGCCCGAC 20
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
TTCATGGTTT CGGAGGCCCG 20
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
GACCGCTTAC CTTGGCATGG 20
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
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-57-
CCTGGGACCA CTGAGGACAG 20
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
GGGACTCACC TTCGTGATGA 20
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
GAACTTCACC ACTGCATGAG 20
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid --
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
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-58-
TCCCAAAGAT GCCCACCTGC 20
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
GCATAATCTG GAAAGGAAGG 20
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
ACATCCTCAC CTGCATTCAC 20
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
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-59-
ACATCCUCAC CTGCAUUCAC 20
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STR.A.NDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
TTTCTTTGGT CTGCAATGGG 20
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
GGCCACTTAC TTTTCTTGTC 20
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
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-60-
CACAGGGACT GGAAAATAAA 20
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
GGGAACCAAC CTGCAAGTAC 20
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
GTCACATCTG AGGGAAATGG 20
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
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-61-
CAGCCTGGCT CACCGCCTTG G 21
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPQLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
TCGCGCTCCC TCTCTCCGGC 20
(2) INFORMATION FOR SEQ ID NO:48
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
CATGGTTTCG GAGGGCGTC 19.
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid - -
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
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-62-
TCCGAAACCA TGAACTTTCT G 21
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
GGTTTCGGAG GCCCGACCG 19
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
CAAGAGAGCA GAAAGTTCAT 20
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
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-63-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
CACCCAAGAG AGCAGAAACT 20
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
TCGTGGGTGC AGCCTGGGAC 20
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
CACCCAAGAC AGCAGAAAG 19
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA -
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
GCACCCAAGA CAGCAGAAAG 20
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
TGCACCCAAG ACAGCAGAAA G 21
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
ATGCACCCAA GACAGCAGAA AG 22
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
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AATGCACCCA AGACAGCAGA AAG 23
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
CAATGCACCC AAGACAGCAG AAAG 24
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
CCAATGCACC CAAGACAGCA GAAAG 25
(2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
TCCAATGCAC CCAAGACAGC AGAAAG 26
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(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
CTCCAATGCA CCCAAGACAG CAGAAAG 27
(2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
GCTCCAATGC ACCCAAGACA GCAGAAAG 28
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
GGCTCCAATG CACCCAAGAC AGCAGAAAG 29
(2) INFORMATION FOR SEQ ID NO:65:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
CACCCAAGAC AGCAGAAA 18
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
CACCCAAGAC AGCAGAA 17
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
CACCCAAGAC AGCAGA 16
(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
CACCCAAGAC AGCAGAAAGT T 21
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA/RNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
CACCCAAGAC AGCAGAAAGT TCAT 24
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
TCGCGCTCCC TCTCTCCGGC 20
(2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear -
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7l:
CATGGTTTCG GAGGGCGTC 19
(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
CAGCCTGGCT CACCGCCTTG G 21
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
TCCGAAACCA TGAACTTTCT G 21
(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
CTTTCTGCTG TCTTGGGTG 19
